Understanding the relationship between sequence, structure and function is an important goal in rational protein engineering. We present our approach how to obtain this understanding for the enzyme class of lipases (E.C. 3.1.1.3). These enzymes are widely used and catalyze the cleavage of waterinsoluble esters. Over the past 30 years, sequence and structure information on lipases accumulated abundantly, highly pure recombinant lipases have become available, and the number of patents and reports on their improvement by protein engineering is steadily increasing.
To obtain a global understanding for the sequencestructurefunction relationship of lipases, we assembled published information on lipases and established the Lipase Engineering Database (LED). By aligning and classifying sequences, superimposing structures and annotating them consistently, the shapes of substrate binding sites were compared and related to biochemical properties. Two important properties, the specificity profile toward scissile fatty acids and enantioselectvity toward chiral alcohols, were investigated by molecular modeling.
To understand the fatty acid chain length profile of lipases, schematic shapes were generated. Residues which mediate chain length specificity were identified and the experimentally observed effect of mutations could be understood.
To investigate the enantioselectivity of lipases toward chiral alcohols, various substrates were docked using molecular dynamics simulations and geometric parameters were extracted, which correlate with experimentally observed enantioselectivities. Thus, comparing and analyzing shape and properties of lipase binding sites and studying enzymesubstrate complexes, intermolecular interactions were identified which mediate specificity and selectivity.